The goal of this project is to apply and develop molecular biological methods that can ensure that vaccines and cell substrates are free from viral, and particularly retroviral, contamination. Three sensitive methods for the detection of reverse transcriptase (RT) that are at least a million-fold more sensitive than conventional RT assays have been published. Although the assays were variously termed PERT (for product-enhanced RT) or Amp-RT, we have chosen to call them by the generic name of PBRT for PCR-based RT. The three assays depend on the same principle: an RNA of known sequence is used as a template with an oligodeoxynucleotide primer in an RT reaction; because the sequence of the RNA is known, the cDNA product can be amplified by the polymerase chain reaction (PCR); the PCR product can be detected by a number of methods. We set up the three PBRT assays at CBER in order to compare their sensitivities, specificities, and reproducibilities. Modifications were made to the assays to eliminate one problem with the assays, viz., their high background signals. We also modified the assay such that RT activities of cellular DNA polymerases were substrantially reduced. Recently, we have adapted the PBRT assay for use with the real time quantitative system, the TaqMan system, for use with the Perkin-Elmer 7700 system. This modified PBRT assay, the TM-PERT assay, is linear over at least 6 orders magnitude and is as sensitive as the original PBRT assays. With this sensitive TM-PERT assay, we have undertaken collaborative studies with several groups at CBER to apply the assay to several regulatory issues. In a study with Kurt Brorson and colleagues (OTRR/DMA/LMDI), we are assessing whether the TM-PERT assay can be used to monitor viral clearance during the preparation of monoclonal antibodies. Currently, methods such electron microscopy and infectivity assays are used by industry. These assays are costly, time-consuming, non-quantitative, and quite variable. The application of the senstive and quantitative TM-PERT assay to follow viral clearance has obvious attractions. Preliminary experiments suggest that the assay will be suitable for this function. In a separate study with Carolyn Wilson, we determined the RT level in porcine factor VIII, a therapeutic used in a small percentage of hemophiliacs when human factor VIII is unsuitable. Because the product is derived from pig cells and because all pig cells have and some may express an endogenous retrovirus (PERV), the potential exists that porcine factor VIII preparations have PERV. All factor VIII lots tested had low levels of PERV as assessed by the TM-PERT assay by us and by RT-PCR by Carolyn Wilson. Fortunately, Dr Wilson has shown that these factor VIII preparations do not contain infectious virus, and thus their continued use is warranted.